The surface to volume ratio increases dramatically when miniaturizing within the nl- and pl-range. It is thus more critical with effective precautions to hinder undesired surface interactions when handling liquid volumes in the lower part of the μl-range such as nl- and pl-volumes compared to larger volumes. Compared to static systems like microtitre wells the problem is far more accentuated for microfluidic systems in which there typically are relatively long transport microconduits, which provide additional contact surfaces that can provide further possibilities for undesired interactions with transported reactants.
Problems with undesired interactions between soluble reactants and functionalized surfaces/reaction areas/reaction zones in microdevices, such as microfluidic devices, have typically been blocked in similar manners as for larger systems. Well known blocking agents are surfactants, inert proteins, such as serum albumin, casein, non-fat dry milk, lactalbumin, gelatin etc., and/or low molecular weight compounds, such as glycine. See for instance U.S. Pat. Nos. 6,341,182 and 6,498,010 (Fitzgerald et al); U.S. Pat. No. 6,613,581 (Wada et al); US 20040115709 (Morozov et al); US 2040115721 (Mao et al); US 20040147045 (Nelson et al); and 20040189311 (Glezer et al). For the similar reasons transport microconduits of microfluidic devices have been modified with coats showing reduced undesired adsorption and denaturation of biologically active molecules (anti-fouling coats/surfaces). See for instance WO 0056810 (Gyros AB), WO 03086960 (Gyros AB), U.S. Pat. No. 6,709,692 (Genset), U.S. Pat. No. 6,236,083 (Caliper), U.S. Pat. No. 6,509,059 (Caliper), etc. In liquid samples containing biological components, there is also a risk that reactants and other components aggregate or otherwise interact in an undesirable manner. These latter problems have typically been counteracted by inclusion of various agents such as detergents and tensides and/or other agents that have surface-active properties.
The inventors have found that many analytes and other reactants used in microfluidic protocols need special precautions in order for a given protocol to reach an acceptable detection limit, precision, recovery etc., and/or to avoid loss of analyte due to underside interactions with inner surfaces. It is attractive to explain at least a part of these difficulties in terms of deficiencies in the surface modification methods and/or the material in which the enclosed microchannel structures are fabricated. The problems encountered typically depend on type of analyte, matrix in which the analyte occurs (serum, plasma, urine, culture supernatant etc.), kind of assay, kind of device including inner surfaces of the device etc. Problematic reactants, in particular analytes, typically are bioorganic molecules that in the worst cases so far found exhibit a biopolymer structure such as protein or polypeptide structure, nucleic acid structure, and/or carry positively and/or negatively charged groups for instance by showing a net positive or net negative charge at the pH used, and/or are relatively hydrophobic. The problems typically are more severe and difficult to overcome the lower the concentration of the analyte is in the undiluted sample, such as a biological fluid. Thus problematic analytes are typically abundant at concentrations ≦10−6 mole/l, such as ≦10−9 mole/l or ≦10−10 mole/l or ≦10−11 mole/l or ≦10−12 mole/l or ≦10−13 mole/l or ≦10−14 mole/l.
The inventors have found that there is a significant risk that an increasing amount of reactant will be lost the longer the analyte sample is retained in the transport microconduit of a dispensation device when dispensing minor aliquots from a larger aliquot to individual microchannel structures of a microfluidic device. This in particular applies to analytical protocols, for instance for aliquots containing the analyte.